Highly Pathogenic Avian Influenza A(H5N1) from Wild Birds, Poultry, and Mammals, Peru

We identified highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in wild birds, poultry, and a lion in Peru during November 2022–February 2023 and markers associated with transmission adaptation and antiviral drug resistance. Continuous genomic surveillance is needed to inform public health measures and avoid mass animal deaths.

We amplified influenza A virus genomes by using a modified protocol (7).We prepared libraries by using the Nextera XT DNA Library Preparation Kit (Illumina, https://www.illumina.com)and sequenced them by using the MiSeq Reagent Kit v3 (600-cycle paired-end) on the MiSeq platform (Illumina).We trimmed raw reads, removed host sequences, and then de novo assembled the filtered reads.We identified the resulting contigs as H5N1 by using a BLASTn search (https://blast.ncbi.nlm.nih.gov).We deposited all obtained sequences in GenBank (accession nos.OQ547312-451).

RESEARCH LETTERS
We identified highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in wild birds, poultry, and a lion in Peru during November 2022-February 2023 and markers associated with transmission adaptation and antiviral drug resistance.Continuous genomic surveillance is needed to inform public health measures and avoid mass animal deaths.strains from North, Central, and South America into different groups within subclade 2.3.4.4b.We identified 6 subclades comprising sequences from 1-5 countries (Venezuela, Colombia, Ecuador, Mexico/ Honduras/Costa Rica/Panama/Colombia, Costa Rica/Panama/Colombia, and Ecuador/Peru/Chile) and 1 sequence from Colombia that did not cluster with other strains from South America.Our results suggest that the strains from South America were not monophyletic and represented 7 independent virus introduction events (Figure ), complementing a previous report (8).
We also compared available amino acid sequences of virus proteins among strains from South America to identify differences among subclades (Appendix Table 2).We identified several amino acid changes that were shared among members of the same subclade (Appendix Table 3).Those changes were consistent with our HA phylogenetic analysis, supporting the hypothesis that independent virus introduction events occurred in South America.
We performed molecular marker analysis to identify specific amino acid mutations associated with HPAI adaptation, transmission, and antiviral drug resistance, such as those in neuraminidase (NA), matrix protein 2, and polymerase acidic protein (9).We identified 21 molecular markers involved in HPAI H5N1 pathogenicity that were present in all analyzed sequences from South America and 7 markers that were found in some sequences (Table ).However, 2 mutations in the polymerase basic 2 protein (Q591K and D701N) associated with mammal adaptation were identified only in sequences from sea lions in Peru and from 1 human case in Chile.The T271A mutation in polymerase basic 2 protein linked to mammal adaptation and S369I and I396M mutations in NA that were observed in the mink outbreak in Spain (2) were not found in sequences from South America.We did not find amino acid mutations related to resistance to the antiviral drugs oseltamivir, zanamivir and peramivir (in NA), amantadine and rimantadine (in matrix protein 2), or baloxavir (in PA).We only Increases replication in mammalian cell and decreases the interferon response V149A Increases virulence and decreases the interferon response in chickens L103F, I106M Increases virulence in mice K55E, K66E, C138F Enhances replication in mammalian cells and decreases the interferon response *Molecular markers of influenza virus strains were identified as previously described (9).HA, hemagglutinin; HPAIV, highly pathogenic avian influenza virus; M1, matrix protein 1; M2, matrix protein 2; NP, nucleoprotein; NS1, nonstructural protein 1. †Only in 2 sequences from pelicans (GISAID [https://www.gisaid.org]accession nos.EPI_ISL_17099964, EPI_ISL_17165223).‡Only in sequences from 2 sea lions in Peru and 1 human case in Chile.§Only in 1 sequence from a wild bird in Peru (GISAID accession no.EPI_ISL_17660074).¶Mutation sequences from Venezuela and Colombia (Choco) have M rather than V. #Only in sequences from Colombia (Choco).
**Only in 1 sequence from a wild bird in Peru (GISAID accession no.EPI_ISL_17777528).
found the H252Y mutation in NA associated with moderately reduced susceptibility to oseltamivir (10).
In conclusion, HPAI H5N1 virus clade 2.3.4.4b was identified in samples collected in Peru from wild birds, poultry, and a lion during November 2022-February 2023.According to phylogenetic analysis, the multiple cluster distribution revealed independent introductions of HPAI H5N1 clade 2.3.4.4b viruses into South America from North and Central America.Four introductions occurred in Colombia, 2 in Ecuador, and 1 in Venezuela/Peru.In addition, strains from Peru were closely related to those from Ecuador and Chile.Finally, we describe the presence of previously reported mutations that might have public health implications because of their associations with increased virulence and virus replication and mammal host adaptation along with reduced susceptibility to oseltamivir.Continuous genomic surveillance is needed to identify markers associated with mammal adaptation and potential human-to-human transmission, to inform public health measures, avoid mass animal deaths, and to protect human populations.

Figure .
Figure.Phylogenetic analysis of highly pathogenic avian influenza A(H5N1) from wild birds, poultry, and mammals, Peru.Maximum-likelihood method was used for phylogeny of 101 hemagglutinin H5 sequences from avian influenza viruses.Red lines indicate clustering of strains from Peru and sequences from this study; bold font indicates the sequences from this study.Dark blue lines indicate other strains from South and North America.Non-goose/Guangdong lineage virus strains from Eurasia were outgroups.Phylogenetic tree was generated and edited with MEGA X software (https://www.megasoftware.net).Sequences were aligned by using the MUSCLE program in the AliView alignment viewer and editor (https://www.ormbunkar.se/aliview).We used general time reversible and gamma distribution models; robustness of tree topology was assessed with 1,000 bootstrap replicates.Only bootstrap values >70% are shown.Scale bar indicates nucleotide substitutions per site.

Table .
Summary of molecular markers identified in influenza virus strains from South America in study of highly pathogenic avian influenza A(H5N1) from wild birds, poultry, and mammals, Peru* M.A.S., C.D.C., V.E., D.P., and Y.T. are military service members or employees of the US government.This work was prepared as part of their official duties.Title 17, U.S.C., §105 provides that copyright protection under this title is not available for any work of the United States Government.Title 17, U.S.C., §101 defines a US government work as a work prepared by a military service member or employee of the US government as part of that person's official duties.The views, findings, and conclusions in this report are those of the authors and do not necessarily represent or reflect the official policy or position of the Department of the Navy, Department of Defense, or the US government.Ethics approval was not necessary for this study because activities were performed as part of a public health response to avian influenza outbreaks by Servicio Nacional de Sanidad Agraria del Peru and the US Naval Medical Research Unit SOUTH.Environmental fecal samples used for sequencing were collected under Directorial Resolution no.392-2018-MINAGRI-SERFOR-SERFOR-DGGSPFFS of the General Directorate of Sustainable Management of Forest and Wildlife Heritage, National Forest and Wildlife Service, Peru, in cooperation with the Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos.